Provided are a technique for easily forming a spheroid by three-dimensionally culturing hepatocytes, and a technique for forming a spheroid having a higher expression level of a transporter MRP2 playing a role of biliary excretion than that of a conventional method. In order to solve the above-described problems, the present inventors have found out a condition under which hepatocytes easily form the spheroid on a nanopillar sheet. More specifically, this is related to a concentration of type I collagen coated onto the NP sheet. Also, they have found out a condition under which an expression level of a gene related to the excretion of the formed spheroid is improved. More specifically, after the spheroid is previously formed, a biological matrix is overlayered thereon.
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1. A method for culturing animal hepatocytes, comprising the steps of:
coating a nanopillar sheet having spaces between nanopillars smaller than equivalent diameters of hepatocytes with a protein able to bind with integrin and comprising an extracellular matrix component containing type I collagen having a type I collagen concentration within a range of 100 pg/mL to 10 ng/mL;
decompressing the nanopillar sheet on which the protein is coated;
seeding hepatocytes, provided in a culture medium, onto the nanopillar sheet on which the protein is coated; and then
replacing the culture medium after a period of time.
2. The method for culturing animal hepatocytes according to
wherein the step of seeding the hepatocytes comprises adhering the hepatocytes on the nanopillar sheet to form a hepatocyte spheroid.
3. The method for culturing animal hepatocytes according to
the culture medium includes insulin and dexamethasone.
4. The method for culturing animal hepatocytes according to
a concentration of the insulin in the culture medium is within a range of 1 nM to 100 nM, and a concentration of the dexamethasone in the culture medium is within a range of 1 nM to 250 nM.
5. The method for culturing animal hepatocytes according to
a seeding density of the hepatocytes in the culture medium is 1×104 to 1×106 cells/ml.
6. The method for culturing animal hepatocytes according to
after replacing the culture medium, a biological matrix is overlayered.
7. The method for culturing animal hepatocytes according to
the biological matrix contains a basement-membrane derived component.
8. The method for culturing animal hepatocytes according to
the hepatocyte spheroid has a diameter in a range of 30 to 100 μm.
9. The method for culturing animal hepatocytes according to
the period of time before which the culture medium is replaced is 4 hours to 24 hours.
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The present invention relates to a method for forming a three-dimensional spheroid which is a hepatocyte mass or a tissue-like structure by culturing animal hepatocytes with using a culture substratum.
A standard process of drug development can be roughly divided into three steps. A first step is, after investing medicinal needs and selecting a disease to be a target of the drug development, to search an effective substance for the disease, and discover (screen) an objective drug candidate. Subsequently, a second step is to verify the effectiveness and safety for the diagnosis and treatment of the disease targeted by the drug candidate through a nonclinical trial and a clinical trial, summarize its evaluation result in a new-drug application, and obtain its approval from a government. And, a third step is to sell the approved drug in a market to be used for medical care. In this drug-discovery process, as seen from a viewpoint such that a cost for its research and development is high, it has been reported that an amount equivalent to 30%, in some cases, 50% of a total research and development cost is spent on animal experiments.
Animal experiments are used in nonclinical trials, some clinical trials, or initial screening. Therefore, under a recent circumstance that a development cost per one item of the new drug almost reaches 100 billion yen, by developing an alternative low-cost test method instead of the animal experiments, a cost reduction effect in suppressing the research and development cost is expected. Further, also from a viewpoint of animal welfare and protection, particularly as seen from a recent trend in EU, reduction of the animal experiments has been already socially such a large stream that cannot be returned. Still further, it has been pointed out that an in vitro alternative method with using cells is advantageous for figuring out an action mechanism of in-vivo kinetics of a drug or a chemical compound, which is difficult to be understood by conventional animal experiments, more particularly, for evaluating metabolism, detoxification, and hepatic duct excretion in a liver, which is one of important indicators (in Non-patent Document 1).
Under such a background, approaches to alternative methods with using cells have been actively taken in the past. However, many methods have a limitation in the prediction of clinical reactions. A reason for this limitation has been considered that, in these culture methods, cells do not have a structure mimicking an actual in-vivo structure (in Non-patent Document 2). Therefore, the construction of a three-dimensional structure closer to a living body and the establishment of an in vitro assay system with using the structure have been required. More particularly, an in vitro assay system with using hepatocytes is highly expected, and an evaluation system for metabolism or bile canalicular excretion in a liver, which is one of important screening indicators, has been required. The bile canaliculus is a space bounded by two or more hepatocytes, and closes an intercellular space by a tight junction to prevent leakage of bile. The liver transports bile and metabolites from the hepatocytes into the bile canaliculi with using an ATP-dependent transporter expressed in a cell membrane. As a technique for measuring such a hepatic duct excretion, a sandwich method is cited (in Non-patent Document 3). However, practical use of the method showing a sufficient performance has not been achieved yet.
As a device for achieving the three-dimensional structure of the cell, a nanopillar cell culture sheet (hereinafter, referred to as a “nanopillar sheet” or a “NP sheet”) is cited (in Patent Document 1). The NP sheet is a scaffolding material for cell culture or tissue culture, which is produced by applying a microfabrication technology such as a nano-imprint technology. A feature of the nanopillar sheet is that, by using an artificially-designed fine three-dimensional structure as the scaffolding material, an effect as a device for three-dimensional culture can be expected. Some reports for the cell culture with using the nanopillar sheet have been made (in Patent Documents 2 and 3).
A conventional sandwich culture method (hereinafter, referred to as SW culture) is a method for a two-dimensional culture by overlayering Type I collagen on a bottom surface of the hepatocyte and a biological matrix on a top surface of the hepatocyte to sandwich the cell by these two types of substances, and has a feature that the metabolites excreted into the hepatic duct can be quantitated. However, in an in-vitro based SW culture, a dynamic range of the measurement is narrow, and therefore, there is a problem that minute variations cannot be measured. It is considered that this is because an essential cell function of the hepatocyte taken outside once from an inside of the living body is decreased, and the number of bile canaliculi formed in a structure reconstructed in a culture dish is smaller than that inside the living body.
Accordingly, by the three-dimensional culture of the cell, resulting in close to an in vivo tissue structure, a trend of solving the problem has been rapidly spread. More specifically, the culture with using the NP sheet has been attempted. However, regarding the cell culture with using the NP sheet, Patent Documents 2 and 3 describe a three-dimensional culture of fat cells or culture of nerve cells. However, they have not considered the culture of the hepatocytes and do not specifically describe the hepatocyte culture. Therefore, if the methods of Patent Documents 2 and 3 are applied to the hepatocyte culture, there is a problem that a desired spheroid is not formed.
From these reasons, a preferred aim of the present invention is to provide a method for generating a three-dimensional hepatocyte spheroid having a structure and a function close to those of a living body with using an NP sheet.
In order to solve the above-described problems, a condition under which the hepatocytes form the spheroid on the nanopillar sheet has been found out. More specifically, Type I collagen is coated on the NP sheet. Also, a condition under which a gene expression level for excretion of the formed spheroid is improved has been found out. More specifically, after previously forming the spheroid, the biological matrix is overlayered thereon.
By applying the present invention, the spheroid which is the hepatocytes having the three-dimensional structure can be easily formed on the nanopillar sheet. In the formed spheroid, expression of a transporter MRP2 playing a role of biliary excretion is higher than that in a conventional method, and the spheroid has biliary excretion capacity closer to that of the living body. By using such a spheroid, measurement with a wider dynamic range capable of measuring a small amount of the excretion can be achieved. By widening the dynamic range of the measurement, the measurement can be applied to pharmacokinetic tests in the process of drug discovery.
101:
NP sheet
102:
cell culture dish
103:
adhesive
104:
Type I collagen solution
105:
decompression container
106:
decompression pump
107:
flushing saline (Phosphate buffered saline (−);
hereinafter, referred to as PBS(−))
108:
culture medium
109:
hepatocyte
110:
hepatocyte spheroid
111:
decompression chamber
112:
carry-in/out part of decompression unit
113:
open/close valve
114:
connection unit
115:
decompression pump
116:
incubator
117:
liquefied carbon dioxide gas tank
118:
deionized water
119:
carry-in part
120:
carry-out part
121:
inner carry-in part
122:
inner carry-out part
123:
storage room
A best mode for carrying out a method for forming a three-dimensional spheroid which is a hepatocyte mass or a tissue-like structure by culturing hepatocytes with using a culture substratum is described in detail as follows.
In Example 1, a step for forming the hepatocyte spheroid on the NP sheet is described.
<Preparation of Hepatocytes>
The preparation of hepatocytes follows an in-situ collagenase perfusion method. The details are as follows. A male Fisher 344 rat (7 to 10 weeks old) is subjected to laparotomy under pentobarbital anesthesia, a catheter is inserted into a portal vein, and a pre-perfusion solution (Hanks' solution without containing Ca2+ and Mg2+ and with containing EGTA) is injected thereinto. At the same time, an inferior vena cava in a lower portion of the liver is incised to release blood. Next, a thoracic cavity is opened, an inferior vena cava entering a right atrium is incised, and the inferior vena cava in the lower portion of the liver is clamped to perform the perfusion. After sufficient blood removal from the liver is confirmed, the perfusion is stopped. A perfusion solution is replaced with a collagenase solution, and the perfusion is performed. In this example, the perfusion is performed with using a Hanks' solution containing 0.05% collagenase. However, the invention is not limited to the solution. After digestion of an intercellular tissue by the collagenase is confirmed, the perfusion is stopped. The liver is cut and separated, is sliced in a cooled Hanks' solution, and is dispersed by pipetting to be cells. Subsequently, undigested tissues are removed by gauze filtration. A cell suspension is centrifuged at 50 G for 1 minute repeatedly several times to remove nonparenchymal cells. Subsequently, with using an isotonic percoll solution, damaged hepatocytes are removed by centrifugation at 500 G for 5 minutes. A survival rate of the obtained hepatocytes is measured by the trypan blue exclusion method, and hepatocytes with the survival rate of 85% or higher are used for the culture. Here, the hepatocytes with the survival rate of 85% or higher are used for the culture. However, it is needless to say that the invention is not necessarily limited to the condition. Also, the preparation of the hepatocytes is not necessarily limited to the in-situ collagenase perfusion method.
Further, human hepatocytes can be also prepared by the same method.
<Preparation of NP Sheet on which Type I Collagen is Coated>
An NP sheet (101) is attached on a bottom surface of a culture dish (102) (
Next, 1 to 1.5 mL of a diluted solution (Type I collagen solution) 104 obtained by diluting the Type I collagen dissolved in a weak acid solution by sterilized water so as to have a predetermined concentration is added to the NP sheet (
Next, in order to completely adsorb the added Type I collagen on the NP sheet, a decompression operation is performed inside a decompression container 105 by a decompression pump 106 (
Finally, the Type I collagen is removed, and PBS(−) 107 is added (
<Culture of Hepatocytes>
The hepatocytes 109 prepared by the in-situ collagenase perfusion method as described above are suspended in a culture medium 108, and are seeded on the NP sheet which is similarly prepared as described above and on which the Type I collagen has been coated (
In Example 2, the effectiveness of the NP hepatocyte spheroid described in the Example 1 is evaluated. Hereinafter, for each evaluation method, an evaluation procedure and an evaluation result are described in this order.
<Immunostaining>
Immunostaining is performed in order to show that the structure of the formed hepatocyte spheroid is close to the structure of the in-vivo liver tissue. Focused molecules are E-cadherin which is a cell-cell (intercellular) adhesion protein and actin which is a cytoskeletal protein. The E-cadherin is one of membrane proteins playing a role of the cell-cell adhesion binding, and is a protein essential for constructing a tissue having a higher dimensional structure by binding cells with each other. Also, the actin is a cytoskeletal protein providing a physical support to cells when the cells adhere to each other to form the higher dimensional structure. The expression of these two proteins is an index essential to show that the tissue is established as the in-vivo tissue.
The procedure of the immunostaining is described below. First, as following the Example 1, the hepatocyte spheroid is formed with using the NP sheet on which the Type I collagen has been coated (hereinafter, referred to as NP normal culture). In this example, from the results of the Example 1, the spheroid is formed by coating 1/106% (W/V) of the Type I collagen on the 2.0 NP that have particularly developed the actin which is a target skeletal protein and the E-cadherin which is a target cell-cell adhesion protein, and have constructed a nearly spherical spheroid. However, the invention is not necessarily limited to this condition. The formed spheroid is washed with the PBS(−) three times, and fixed. While the spheroid is fixed for 15 minutes with using 4% paraformaldehyde in this example, the invention is not necessarily limited to this method. The spheroid is washed with the PBS(−) three times, and is dipped in a nonionic surfactant for 5 minutes to destroy the cell membrane. While 0.5% Triton X is used in this example, the invention is not necessarily limited to this substance. After washing the spheroid with the PBS(−) three times, blocking is performed. While the blocking is performed by dipping the spheroid in 1% FBS (Fetal Bovine Serum) for 30 minutes in this example, the invention is not necessarily limited to this method. Then, a primary antibody is acted. While a monoclonal rabbit anti-human E-cadherin antibody is acted in this example, the immune animal species is not limited to a rabbit, and may be also, for example, a mouse or a goat. Also, while action temperature and time for the primary antibody are at 4° C. for one day and night in this example, the invention is not limited to them. Subsequently, after washing the spheroid with the PBS(−) containing a nonionic surfactant, it is washed only with the PBS(−). While a substance of 0.05% Tween20/PBS(−) is used in this example, the invention is not limited to this substance. Then, a fluorescein-added (labeled) secondary antibody is acted. While an FITC-added anti-rabbit IgG antibody is acted in this example, the immune animal species is not limited to a rabbit, and may be also, for example, a mouse or a goat. Further, while action temperature and time is at 37° C. for one hour in this example, the invention is not limited to this method. Subsequently, the washing is performed with using the same method. Next, actin fibers are stained. While rhodamine-added phalloidin is acted at a room temperature for 5 minutes in this example, action time is not limited to this. The spheroid is washed with the PBS(−) once, and nuclear staining is performed. While “Hoechst33342” is acted at the room temperature for 5 minutes in this example, the invention is not limited to this method. Finally, the spheroid is washed with Milli-Q water, and is sealed.
Here, for comparison, immunostaining is performed to the hepatocytes obtained by conventional plane culture to verify the expression levels of the E-cadherin and the actin. The conventional plane culture is performed as follows. The hepatocytes are suspended and seeded in the above-mentioned culture medium at a density of 1×105 cells/ml on a commercially-available collagen-coated culture dish. After passing 18 hours or longer from the seeding, a first culture-medium replacement is performed, and then, the culture medium is replaced every 24 hours. The culture medium used for the culture after passing 18 hours from the seeding is as described above (hereinafter, referred to as plane culture). To cells obtained by such a plane culture, the immunostaining is performed by the method as described above.
Similarly, for comparison, the immunostaining is performed also to a frozen liver tissue slice to verify the expression levels of the E-cadherin and the actin. A method for preparing the frozen slice is performed as follows. The method is the same as that in the Example 1 up to the step of, after the laparotomy of the rat, removing the blood with using the pre-perfusate. Then, a part of the liver is cut off, an embedding agent is added thereto, and the liver is frozen in a freezer at −80° C. With using the frozen sample, the slice is produced in a freezing microtome method. The immunostaining is performed to such an obtained frozen slice by the above-described method.
<Ammonia Metabolic Capacity Measurement>
Next, in order to verify the function of the spheroid having the structure close to that of the living body, ammonia metabolic capacity which is an important function of the liver is measured.
Inside the living body, ammonia is mainly produced in the intestine and kidney and is carried to the liver by the bloodstream, and then, the liver converts the ammonia into urea with using the urea cycle. In order to measure the ammonia metabolic capacity of such a liver, a commercially-available dedicated kit is used. First, as following the above-described method, the hepatocyte spheroid is formed by the NP normal culture with using the 2.0 NP sheet. For comparison, the culture is performed as following the above-described plane culture method. In both culture, the medium is replaced with an ammonia culture medium in 72 hours after the start of the culture, and a part of the culture medium is sampled. After the culture for 24 hours from the replacement of the culture medium, the culture medium is similarly sampled. For each sampling liquid, a sample is prepared as following the descriptions of the kit. The absorbance at 630 nm is measured to calculate an amount of ammonia reduction (metabolism) over the 24 hours. Note that, with using a sample whose ammonia concentration is already known, a calibration curve is previously required. Meanwhile, for measuring the number of cells at the sampling, the cells are solubilized by SDS, and then, “Hoechst332582” is added thereto, and its fluorescence intensity is measured to obtain the number (excitation wavelength: 355 nm, fluorescence wavelength: 460 nm). While the commercially-available kit is employed in this example, any method is possible.
From a result of the measurement of the ammonia metabolic capacity of the spheroid having the three-dimensional structure reconstructed with using the NP sheet as described above, the capacity in the NP spheroid is dominantly higher than that of the conventional plane culture, and therefore, it is found out that the ammonia metabolism is actively boosted (
<Study on Bile Canalicular Excretion>
In order to verify the liver function of the spheroid formed with using the NP, an excretion capacity to the bile canaliculi which is one of the important functions of the liver is measured. In this example, the presence/absence of the excretion to the bile duct is verified by dosing 5-carboxy-2′,7′-dichlorofluorescein diacetate (CDFDA) which is a substrate of the MRP2 which is a protein localized on the cell membrane forming the bile canaliculi. When the MRP2 has the activity, the CDFDA is excreted to the bile duct as 5-carboxy-2′,7′-dichlorofluorescein (CDF).
A method of the measurement is performed as follows. First, as following the above-described method, the hepatocyte spheroid is formed by the NP normal culture with using the 2.0 NP sheet. For comparison, the sandwich culture (hereinafter, referred to as SW culture) which has advanced in the quantitative measurement of the biliary excretion is employed. The SW culture is performed as follows. The hepatocytes are suspended and seeded in the above-described culture medium at a density of 1×105 cells/ml on a commercially-available collagen-coated culture dish. The culture is started, the first culture-medium replacement is performed in 4 hours, the culture medium is replaced in 24 hours with a culture medium containing a biological matrix (hereinafter, referred to as biological-matrix culture medium), and then, the culture medium is replaced every 24 hours as following the above-described method for the culture (see
As a result of the measurement, at the time of 24 hours, in both of the NP spheroid culture and the SW culture, an aspect that the uptake CDFDA is accumulated inside the cells is observed (in
<Real-Time PCR>
In order to verify that the formed spheroid maintains the high-dimensional structure as that of the liver tissue or has a liver-specific function, quantitative gene-expression analysis is performed with using the real-time PCR method. An expression level of a target gene of a hepatocyte obtained immediately after its collection is measured (Table 1), and, with taking the expression level as 1, an expression level under each culture condition is calculated, so that the expression levels are compared with each other.
TABLE 1
Gene
Size (bp)
Catalog ID
TBP
153
Rn01455648_ml
E-cadherin
105
Rn00580109_ml
MRP2
60
Rn00563231_ml
Claudin 3
90
Rn00581751_sl
P450-3A3
113
Rn01640761_gH
Albumin
95
Rn00592480_ml
The expression levels are compared among three types of culture conditions (
A scheme of the experiment is shown in
As a result of the experiment, under the condition of 2.0NP+M that the biological matrix is overlayered on the NP spheroid (
A configuration of the culture apparatus achieving the automation of the sequence of culture steps described in the Example 1 and its automatic culture steps are described below.
Also, by previously combining a plurality of conditions as the input conditions and storing them in a memory, the inputting can be simplified. The plurality of conditions are stored, and a condition under which the step is to be operated may be selected via the display unit.
The inputted information is transmitted to a control unit, and is stored in a memory or others. Based on the inputted information, a sequence of commands are invoked from the memory, so that the culture step is started. Note that an operation control for the culture step is collectively managed in a central processor mounted on the control unit. All steps are performed inside a clean room.
Further, a bar code, an RFID tag, or others is mounted on each culture dish, and a device for reading them is provided on each unit for executing the step, so that each step of the culture dish can be managed by electronic communications in the control unit.
The NP sheet is carried by a robotic arm, a belt conveyor, or other methods. Also, in a case that a part of the steps is independent in a separate apparatus, the culture dish may be carried in or out by an operator.
The culture dish on which the NP sheet is attached is first transferred to the type I collagen injection unit, and then, is put inside thereto from a carry-in/out part. A nozzle for injecting the Type I collagen into the culture dish is provided in the Type I collagen injection unit. An injection amount of the Type I collagen is previously stored in the memory, and an amount covering a projection tip portion of the NP sheet is preferable.
The culture dish to which the Type I collagen has been injected is carried out from the carry-in/out part, and is subsequently transferred to the decompression unit (
After the washing operation is finished, the culture dish is transferred to the cell suspension injection unit. In the cell suspension injection unit, a cell suspension and a nozzle for injecting the cell suspension into the culture dish are provided. A predetermined amount previously stored in the memory is injected. Also, the cell suspension injection unit is configured such that its stage vibrates vertically and horizontally so as to evenly seed the injected cell suspension onto the culture dish. Further, a heat insulating material is provided in the cell suspension injection unit, and its temperature can be controlled by the control unit.
The culture dish to which the cell suspension has been injected (hereinafter, referred to as cultured cells) is subsequently transferred to the culture unit (
In the culture-medium replacement unit, a suction device having a suction nozzle at its tip portion and a culture medium having an injection nozzle at its tip portion are provided. The culture-medium replacement unit includes some chambers whose temperatures are controlled by the control unit, and the cultured cells are transferred to a culture-medium replacement chamber having a predetermined temperature in accordance with the intended use for the culture-medium replacement. The tip portion of each nozzle has exchangeable means for preventing contamination. The cultured cells having finished the culture-medium replacement are transferred to the culture unit again for the culture.
Each of the culture and the culture-medium replacement is performed a predetermined number of times for predetermined time, so that a desired spheroid can be obtained.
A monolayer culture on a two-dimensional plane has been a main stream of the cell culture. However, an in-vivo environment is three dimensional, and therefore, it is essential to form a cell culture system mimicking the environment and a cell group having a three-dimensional structure. In the present invention, by defining the concentration of the Type I collagen coated on the NP sheet, the spheroid which is the cell mass having such a three-dimensional structure can be easily formed. Besides, the spheroid formed by the present invention is a spheroid expressing the function close to that of the in-vivo liver tissue having the biliary excretion capacity, and its utility value in the drug discovery screening field is high.
Takahashi, Ryosuke, Hisada, Akiko, Sonoda, Hiroshi
| Patent | Priority | Assignee | Title |
| 11041145, | Aug 25 2016 | PHILIP MORRIS PRODUCTS S A | Cell culture |
| 11981926, | Aug 25 2016 | PHILIP MORRIS PRODUCTS S.A. | Cell culture |
| Patent | Priority | Assignee | Title |
| 20040125266, | |||
| 20060183222, | |||
| 20070299537, | |||
| 20080057578, | |||
| 20080215073, | |||
| CN101050418, | |||
| JP2004170935, | |||
| JP2006223197, | |||
| JP2008199962, | |||
| JP200854566, | |||
| JP7274952, | |||
| WO2005047496, |
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